Rapid and Specific Detection of Active SARS-CoV-2 With CRISPR/Cas12a.

Xinyi Liu,Xin Wang,Yifan Song,Lina Wu,Xiaodong Ma,Xingxu Huang,Ming Liu,Yanhua Li,Peixiang Ma,Xinjie Wang,Benyuan Yu

doi:10.3389/fmicb.2021.820698

Abstract

Rapid and sensitive nucleic acid detection of SARS-CoV-2 has contributed to the clinical diagnosis and control of COVID-19. Although detection of virus genomic RNA (gRNA) has been commonly used in clinical diagnosis, SARS-CoV-2 gRNA detection could not discriminate between active infectious virus with remnant viral RNA. In contrast to genomic RNA, subgenomic RNAs (sgRNAs) are only produced when the virus is actively replicating and transcription, detection of sgRNA could be an indication to evaluate infectivity. CRISPR/Cas-based nucleic acid detection methods have been considered potential diagnostic tools due to their intrinsic sensitivity, specificity and simplicity. In this study, to specifically detect active virus replication, we developed a CRISPR-based active SARS-CoV-2 (CRISPR-actCoV) detection strategy by detecting sgRNAs of SARS-CoV-2. CRISPR-actCoV with CRISPR Cas12a-assisted fluorescence reporter system enables detection of sgRNAs at 10 copies in 35 min with high specificity and can be read out with naked eyes. Further, we performed CRISPR-actCoV mediated sgRNA detection in 30 SARS-CoV-2 potentially infected clinical samples, and 21 samples were SARS-CoV-2 sgRNA positive. A quantitative RT-PCR assay was also performed to detect gRNA of SARS-CoV-2 in parallel. Among the 30 clinical samples, 27 samples were gRNA positive. Taken together, CRISPR-actCoV provides an alternative for rapid and accurate detection of active SARS-CoV-2 and has great significance in better response of coronavirus causing epidemic disease.

Highlights

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19 respiratory disease, has spread worldwide and affected global health and the world economy
Amplification and Verification of SARS-CoV-2 Subgenome in a Diagnosed Clinical Sample subgenomic RNAs (sgRNAs) of SARS-CoV-2 are only produced during viral replication and are poorly packaged into mature virions, they have been proposed to represent a potential marker for active infection and viral replication (Immergluck et al, 2021)
Virus sgRNAs were amplified by RT-RPA to obtain enough DNA substrates using specific primers, and the amplicons were subjected to CRISPRCas12a detection assays

Summary

Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19 respiratory disease, has spread worldwide and affected global health and the world economy. Rapid and accurate nucleic acid detection of SARS-CoV-2 has contributed to the early diagnosis and treatment of COVID-19 (Chu et al, 2020; Orive et al, 2020; Seo et al, 2020; Wang et al, 2020c). Viral gRNA might still be detectable in months after clinical recovery (Xiao et al, 2020), and detection of gRNA does not prove the presence of active viral replication or infectious virus. It can not distinguish replicable virus from remnant viral RNA or laboratory-generated non-hazardous nucleic acids (Robinson-McCarthy et al, 2021)

Methods

Results

Conclusion